Method for adding an apparent non-signal line to a rapid diagnostic assay

ABSTRACT

A test device and method for determining the presence or absence of one or more analytes in a fluid sample, the test device including a support or member bearing a mark thereon, and a matrix or member containing a capture zone. In operation, an observation area in the test device becomes transparent, thereby allowing the user to view a mark that is present on a support that is disposed beneath the observation area. Typically, the mark on the underlying support is configured as a minus (−) sign. In the absence of analyte in the sample, the test device presents a negative result as a minus (−) signal. In the presence of analyte in the sample, however, the mark operates in concert with a perpendicular test line on the observation area to present a positive result as a plus (+) signal that is visible to the user.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part of U.S. applicationSer. No. 09/950,366; filed Sep. 10, 2001, now U.S. Pat. No. 6,855,561,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

The present invention relates generally to devices and methods fordetection of analytes in test samples. More specifically, the presentinvention provides solid phase test devices and methods that combine aninternal indicator on the test with an external mark located on asupport.

Various analytical procedures and devices are commonly employed indetection assays to determine the presence and/or amount of substance ofinterest or clinical significance which may be present in biological ornon-biological fluids. Such substances are generally termed “analytes”and can include antibodies, antigens, drugs, or hormones.

The present invention includes, but is not limited to, lateral flowchromatography assay formats. Generally, these assays have an extendedbase layer on which a differentiation can be made between a sampleapplication region and an evaluation region. In typical use, the sampleis applied to the sample application region, flows along a liquidtransport path which runs parallel to the base layer, and then flowsinto the evaluation region. A capture reagent is present in theevaluation region, and the captured analyte can be detected by a varietyof protocols to detect visible moieties associated with the capturedanalyte. For example, the assay may produce a visual signal, such ascolor change, fluorescence, luminescence, and the like, when indicatingthe presence or absence of an analyte in a biological sample.

Vertical flow devices and assays are also contemplated herein.Generally, these assays, similar to those described in U.S. Pat. No.4,632,901, involve the introduction of a liquid sample to a device andallowing the fluid sample to pass through one or more layers to generatea result. Results, similar to the assays described above and below, maybe in the form a visual signal.

Optimally, such test devices will provide a characteristic signal whenthe analyte is present in a sample, and a different signal when theanalyte is absent from a sample. Most typically, the test device willdisplay a “plus” (+) signal in the presence of analyte, and a “minus”(−) signal in the absence of analyte. The plus/minus test result formathas enjoyed enthusiastic customer response and wide commercial success.

Test devices of this kind are well known in the art, and play animportant role in areas such as clinical chemistry. They are used byskilled clinicians and lay person alike. Thus, there is a strong impetusto provide devices that are simple and reliable. Desirably, the assaysare single-step devices wherein the user need only apply the sampleprior to viewing the result. Single-step devices obviate the necessityof performing complicated and time consuming processing steps that mayintroduce errors in the end result.

Examples of such assays include pregnancy tests, ovulation tests,various urine, saliva, spinal, and blood tests, as well as otherclinical or diagnostic assays.

Test devices typically use materials that specifically bind with ananalyte of interest. A homologous pair of specific binding pair members(“sbp members”), usually an immunological pair comprising a ligand and areceptor (antiligand), is involved, wherein one of the sbp members islabeled with a label that provides a detectable signal. The immunoassaymethodology results in a distribution of the signal label between signallabel bound in a complex of the sbp members and unbound signal label.The differentiation between bound and unbound signal label can be aresult of physical separation of bound from unbound signal label ormodulation of the detectable signal between bound and unbound signallabel.

In developing an assay device, there are many considerations. Oneconsideration is to provide substantial differentiation between theobserved signal resulting from signal label when bound as compared tounbound. Another consideration is the ease with which the observedsignal can be detected and serve to differentiate between the presenceor absence of analyte of interest. Other factors include the precisionwith which the test devices must be manufactured. In factoring thisconsideration it is important to include registration or indexingcapabilities in vertical flow test devices of the present invention.These capabilities, as described in detail below, are important fortesting accuracy, reproducibility and ease of use and reading results.Therefore, in developing an assay that can be used by untrainedpersonnel, such as assays to be performed in the home, medical officesand the like, the technique for performing the assay should be simple,and the method of manufacturing the assay should be straightforward.

Plus/Minus Assays

Of particular interest to the present invention are test devices of thetype described in U.S. Pat. No. 5,145,789 to Corti et al., thedisclosure of which is incorporated herein by reference. Corti et al.discuss a built-in positive control to indicate successful operation ofa pregnancy test device. The positive control is envisaged as ahorizontal tract that always stains, independent of the presence of hCGin the urine, and is described as an area on a membrane that containsimmobilized hCG. Regardless of whether hCG is present in the biologicalsample, it is intended that during operation, the upstream mobilelabeled hCG binding reagents will always bind to the immobilized hCG,thereby forming a horizontal line, or minus sign, in the reading area.

A similar approach for providing a minus sign in a test device isdescribed in U.S. Pat. Nos. 4,916,056, 5,008,080 and 5,160,701 to Brown,III et al., the disclosures of which are incorporated herein byreference. As illustrated, the positive control is formed by providing abinding substance within the test strip matrix, and is formed in theshape of a rectangular bar, or minus sign. The binding substance of theminus sign is intended to bind the labeled material regardless of thepresence or absence of the analyte of interest in the test sample.

Another approach for providing a positive control in a test device isdescribed in EP Patent Publication No. 0 249 418 to Graham, Jr., thedisclosure of which is incorporated herein by reference. As described,the control zone has anti-human IgG or IgM immobilized thereon, fornonspecifically capturing human immunoglobulin ubiquitously present inall similar human aqueous samples. The immobilized antibody is intendedto provide a signal in a “minus” pattern, regardless of the presence orabsence of the analyte of interest in the test sample.

Osikowicz et al., in U.S. Pat. No. 5,075,078, describe yet anotherapproach for providing a positive control in a plus/minus test device.The positive control is disposed on a test strip in a rectangular barconfiguration. The control bar is oriented on the strip so that it liesneither perpendicular nor parallel to the direction of fluid flow, butrather lies at an intermediate orientation, i.e., at a 45 degree angle.

Still yet another approach for providing a positive control in a testdevice is provided in U.S. Pat. No. 5,401,667 to Koike. As described,the test device provides a plus/minus format, but considers alternativegeometric symbols as well. A portion of the chromatographic medium isremoved, or otherwise partially blocked, thereby affecting the flow pathof the liquid. It is suggested that this modification enhances thesignal of the device.

Wong et al., in EP Patent No. 0 260 965, describe another test devicethat utilizes the plus/minus format. Wong et al. discuss a multiple-stepdiagnostic assay with a horizontal positive control line sprayed onto atest membrane.

The previous methods discussed above accomplish the “appearance” of aminus sign (−) by placing an indicator (positive control) lineperpendicular to the test line, directly onto the test strip. Typically,the control line develops with any sample flow, while the test linedevelops only with a positive sample flow. Thus, the previous assaysinvolve a control mechanism inherent to the matrix membrane test strip,and require a specific manufacturing step to apply the control line tothe strip.

Other previous devices display a printed minus sign positioned on thematrix and across the test line. These devices typically incorporate apositive control line downstream from, and parallel to, the test line.Such devices are limited as the test strips may present a line that isvisible before the sample is added.

Previous methods are further disadvantaged as the additionalmanufacturing step involves a difficult placement procedure to orientthe perpendicular line directly in the center of the viewing window.Whether the perpendicular line is a printed minus sign, or areagent-based control line, this approach is particularly ill suited forcertain matrix construction procedures, including web processing methodsthat involve a continuous flow or continuous roll application approach.

Therefore, it would be desirable to provide a test device that does notrequire this extra processing step of depositing a perpendicular lineonto the test strip, or does not leave a line that is visible before thesample is added to the device. This invention fulfills these and otherneeds.

Transparent Membranes

The use of transparent test strips in diagnostic assays is known in theart. In U.S. Pat. No. 4,824,640, Hildebrand et al. discuss a transparentreagent carrier layer suitable for evaluation by transmissionphotometry. As described, the transparent nature of the film of plasticprovides a suitable carrier material as compared to opaque films.

The use of a transparent test strip is also discussed in U.S. Pat. No.5,110,550 to Schlipfenbacher et al. As described, this test deviceincludes a white non-transparent covering layer situated above acolor-forming layer. During operation of the test device, the coveringlayer becomes transparent in the moist state. Through the transparentcovering layer, the user is then able to observe any reaction occurringin the color-forming layer below.

The use of a clearing agent in an immunochromatographic assay isdiscussed in U.S. Pat. No. 6,165,798 to Brooks. As described, the teststrip membrane is rendered transparent by wetting the membrane with aclearing agent, thus reducing the amount of light scattered by themembrane fibers.

In U.S. Pat. No. 6,187,268, Albarella et al. describe a transparent flowthrough membrane for use in test devices, but do not suggest a controlfeature to indicate a positive or negative test result. The membranedescribed in Albarella et al. is not configured to become transparentonly when wet. The membrane is transparent whether wet or dry.

While conceivably workable in some circumstances, the previous detectionsystems that employ transparent membranes are of limited utility. Thereis no teaching or suggestion in current art for a test device with atransparent membrane that utilizes a control feature to indicate apositive or negative test result as provided by a mark on the underlyingsupport.

In view of the foregoing, there remains a need in the art for a simple,efficient method for adding a positive control to a solid phase assaythat does not require the manufacturing step of fixing a positivecontrol binding member to the assay test strip, and that does not leavea substantially visible signal before the sample is added to the device.It would further be desirable to achieve improved test device formatsthat incorporate transparent membranes as part of a control or displayfeature.

Additionally, the assay of the present invention should overcome thedisadvantages described above in connection with the previous testdevice systems.

BRIEF SUMMARY OF THE INVENTION

The present invention includes devices, methods, and kits for visuallydetecting the presence or absence of an analyte in a sample. Forexample, the present invention provides a test device for determiningthe presence or absence of an analyte in a fluid sample. In a particularembodiment, testing devices are contemplated that are capable ofmeasuring multiple analytes in a given sample. The test device may be avertical flow test device, or a lateral flow test device, or any othersolid phase test device that comprises a matrix disposed above asupport, e.g., a dipstick-type testing device.

In one embodiment, the matrix defines an axial flow path, and typicallycontains three zones. First, a sample receiving zone is located at anupstream end of the flow path. The test sample suspected of containingan analyte of interest is applied to the sample receiving zone. Second,a label zone is located on the flow path downstream from the samplereceiving zone. As the test sample flows through the label zone, itcontacts a detectable mobile labeled reagent. This labeled reagent isspecific for, and binds to, the analyte of interest. Third, anobservation area is located at the downstream end of the flow path. Acapture zone is situated in the area, and contains immobilized capturereagents that are specific for, and bind with, the analyte of interest.Generally, the binding is achieved by either a direct or an indirectbinding approach. Optionally, an absorbent zone is disposed downstreamof the observation area.

The sample fluid travels from the sample receiving zone, through thelabel zone, and then across the observation area and capture zone. Theobservation area, now moistened from the sample fluid, becomestransparent. The user can then view a mark displayed on the underlyingsupport.

In another embodiment, a test device is provided useful for determiningthe presence or absence of an analyte in a fluid sample, the test devicecomposed of a matrix comprising a porous first member comprising adefined capture zone and an observation area, and a second memberdisposed below and contacting said first member. In one aspect, thecapture zone contains an immobilized capture reagent that restrains theanalyte, and the first member is comprised of a material that is opaquein a dry state and transparent in a moist state. In another aspect, thesecond member bears one or more marks thereon, and is comprised of aporous material. The second member generally allows fluid flow throughthe first member when a fluid sample is introduced. And, the mark on thesecond member becomes visible through the observation area on the firstmember when the first member is in a moist state. The mark may have ashape such that, in the presence or absence of the analyte in thesample, a first symbol may be displayed to represent a negative result,and the capture zone may have a shape such that, taken together with thefirst symbol in the presence of the analyte in the sample, a secondsymbol is formed to represent a positive result.

In another embodiment, methods of manufacturing and using the testingdevices are provided. In manufacturing vertical flow assays, it isimportant to include registration or indexing capabilities in verticalflow test devices. These capabilities, as described in detail below, areimportant for testing accuracy, reproducibility and ease of use andreading results.

The present invention is simple, effective, and solves the problems ofprevious devices and methods, by providing a test device with atransparent membrane and an underlying support that bears a mark.

This invention utilizes a property of nitrocellulose, or a similarmembrane, in that it becomes transparent when it is wet. By placing adetectable line or symbol directly underneath the nitrocellulose capturezone, that marking will be mostly invisible until a liquid sample isadded. When the membrane becomes wet, the markings underneath it becomeinstantaneously visible and appear to be a part of the displayedresults.

The invention described herein allows the mark to be placed in aprecisely controlled location within the window and can be printeddirectly on the plastic housing or on an intermediate member disposedbetween the housing and the test strip. This allows for the actual teststrip to be processed continuously in that there is no need tomanufacture a control line or minus symbol that is perpendicular to theprocessing flow. This feature is particularly desirable in webprocessing procedures. With appropriate indexing of the printingequipment, the mark can also be printed on the transparent backingmaterial that may be used to support the membrane on which the assaytakes place.

The invention does not require any chemical interaction between a labelcomponent and a capture component to produce the appearance of a line orsymbol in any shape or color. The invention imparts a very distinct lookand operability characteristic to a lateral flow device.

The present invention achieves the “appearance” of a minus sign (−) byplacing an indicator mark perpendicular to the test line, directly onthe underlying support. The mark typically manifests with any sampleflow, while the test line develops only with a positive sample flow. Afurther advantage of the present invention is the avoidance of a linethat is substantially visible before the sample is added to the device.

Other objects and advantages will become apparent from the followingdetailed description taken in conjunction with the drawings in which:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an exploded perspective view of a test device of the presentinvention.

FIG. 2 is an exploded perspective view of a test device of the presentinvention showing a negative test result.

FIG. 3 is an exploded perspective view of a test device of the presentinvention showing a positive test result.

FIG. 4 is an exploded perspective view of a test device of the presentinvention showing a positive test result, and a procedural control line.

FIG. 5 shows a top view of a vertical flow device, looking down into thechamber into which the sample and other components are poured.

FIG. 6 shows a top view of a porous positive control membrane support ina vertical flow device.

FIG. 7 shows a top view of a filter/membrane including a capture/testzone in a vertical flow device.

FIG. 8 shows an illustration of a negative test result in a verticalflow device of the present invention. The positive control line isvisible but the dotted invisible test line is not formed.

FIG. 9 shows an illustration of a positive test result in a verticalflow device of the present invention. Both the positive control line andthe test line are visible.

FIG. 10 shows a side view of a vertical flow device of the presentinvention.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. If a definition set forth inthis section is contrary to or otherwise inconsistent with a definitionset forth in the patents, applications, published applications and otherpublications that are herein incorporated by reference, the definitionset forth in this section prevails over the definition that isincorporated herein by reference. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, the preferred methods andmaterials are now described. All publications mentioned herein areincorporated herein by reference to disclose and describe the methodsand/or materials in connection with which the publications are cited.

In describing the various aspects of the present invention, a number ofterms will be generally defined or abbreviated as follows.

Definitions

As used herein, “a” or “an” means “at least one” or “one or more.”

“Fluid sample” refers to a material suspected of containing the analyteof interest. The fluid sample can be used as obtained directly from thesource or following a pretreatment so as to modify its character. Suchsamples can include human, animal or man-made samples. The sample can beprepared in any convenient medium which does not interfere with theassay. Typically, the sample is an aqueous solution or biological fluidas described in more detail below.

The fluid sample can be derived from any source, such as a physiologicalfluid, including blood, serum, plasma, saliva, sputum, ocular lensfluid, sweat, urine, milk, ascites fluid, mucous, synovial fluid,peritoneal fluid, transdermal exudates, pharyngeal exudates,bronchoalveolar lavage, tracheal aspirations, cerebrospinal fluid,semen, cervical mucus, vaginal or urethral secretions, amniotic fluid,and the like. Herein, fluid homogenates of cellular tissues such as, forexample, hair, skin and nail scrapings, meat extracts and skins offruits and nuts are also considered biological fluids. Pretreatment mayinvolve preparing plasma from blood, diluting viscous fluids, and thelike. Methods of treatment can involve filtration, distillation,separation, concentration, inactivation of interfering components, andthe addition of reagents. Besides physiological fluids, other samplescan be used such as water, food products, soil extracts, and the likefor the performance of industrial, environmental, or food productionassays as well as diagnostic assays. In addition, a solid materialsuspected of containing the analyte can be used as the test sample onceit is modified to form a liquid medium or to release the analyte. Theselection and pretreatment of biological, industrial, and environmentalsamples prior to testing is well known in the art and need not bedescribed further.

“Specific pair binding member” refers to a member of a specific bindingpair, i.e., two different molecules wherein one of the moleculesspecifically binds with the second molecule through chemical or physicalmeans. The two molecules are related in the sense that their bindingwith each other is such that they are capable of distinguishing theirbinding partner from other assay constituents having similarcharacteristics. The members of the specific binding pair are referredto as ligand and receptor (antiligand), sbp member and sbp partner, andthe like. A molecule may also be a sbp member for an aggregation ofmolecules; for example an antibody raised against an immune complex of asecond antibody and its corresponding antigen may be considered to be ansbp member for the immune complex.

In addition to antigen and antibody specific binding pair members, otherspecific binding pairs include, as examples without limitation, biotinand avidin, carbohydrates and lectins, complementary nucleotidesequences, complementary peptide sequences, effector and receptormolecules, enzyme cofactors and enzymes, enzyme inhibitors and enzymes,a peptide sequence and an antibody specific for the sequence or theentire protein, polymeric acids and bases, dyes and protein binders,peptides and specific protein binders (e.g., ribonuclease, S-peptide andribonuclease S-protein), and the like. Furthermore, specific bindingpairs can include members that are analogs of the original specificbinding member, for example an analyte-analog or a specific bindingmember made by recombinant techniques or molecular engineering.

An sbp member is analogous to another sbp member if they are bothcapable of binding to another identical complementary sbp member. Suchan sbp member may, for example, be either a ligand or a receptor thathas been modified by the replacement of at least one hydrogen atom by agroup to provide, for example, a labeled ligand or labeled receptor. Thesbp members can be analogous to or complementary to the analyte or to ansbp member that is complementary to the analyte.

If the specific binding member is an immunoreactant it can be, forexample, an antibody, antigen, hapten, or complex thereof. If anantibody is used, it can be a monoclonal or polyclonal antibody, arecombinant protein or antibody, a chimeric antibody, a mixture(s) orfragment(s) thereof, as well as a mixture of an antibody and otherspecific binding members. The details of the preparation of suchantibodies and their suitability for use as specific binding members arewell-known to those skilled in the art.

When an immunoreactive specific binding member is attached to thechromatographic material of the present invention, the device isreferred to as an “immunochromatograph”, and the corresponding method ofanalysis is referred to as “immunochromatography”. Immunochromatography,as used herein, encompasses both sandwich and competitive immunoassaytechniques.

“Ligand” refers to any compound for which a receptor naturally exists orcan be prepared.

“Receptor” refers to any compound or composition capable of recognizinga particular spatial or polar orientation of a molecule, e.g., epitopicor determinant site. Illustrative receptors include: antibodies,enzymes, thyroxine binding globulin, intrinsic factor, lectins, nucleicacids, protein A, complement, complement Clq, and the like. Receptorsare also referred to as antiligands.

“Antigen” shall mean any compound capable of binding to an antibody, oragainst which antibodies can be raised.

“Antibody” refers to a polypeptide substantially encoded by animmunoglobulin gene or immunoglobulin genes, or fragments thereof. Therecognized immunoglobulin genes include the kappa, lambda, alpha, gamma,delta, epsilon, and mu constant regions, as well as myriadimmunoglobulin variable region genes. Light chains are classified aseither kappa or lambda. Heavy chains are classified as gamma, mu, alpha,delta, or epsilon, which in turn define the immunoglobulin classes, IgG,IgM, IgA, IgD, and IgE, respectively. Typically, an antibody is animmunoglobulin having an area on its surface or in a cavity thatspecifically binds to and is thereby defined as complementary with aparticular spatial and polar organization of another molecule. Theantibody can be polyclonal or monoclonal. Antibodies may include acomplete immunoglobulin or fragments thereof. Fragments thereof mayinclude Fab, Fv and F(ab′)2, Fab′, and the like. Antibodies may alsoinclude chimeric antibodies made by recombinant methods.

“Analyte” or “analyte of interest” refers to the compound or compositionto be detected or measured and which has at least one epitope or bindingsite. The analyte can be any substance for which there exists anaturally occurring analyte specific binding member or for which ananalyte-specific binding member can be prepared. e.g., carbohydrate andlectin, hormone and receptor, complementary nucleic acids, and the like.Further, possible analytes include virtually any compound, composition,aggregation, or other substance which may be immunologically detected.That is, the analyte, or portion thereof, will be antigenic or haptenichaving at least one determinant site, or will be a member of anaturally-occurring binding pair.

Analytes include, but are not limited to, toxins, organic compounds,proteins, peptides, microorganisms, bacteria, viruses, amino acids,nucleic acids, carbohydrates, hormones, steroids, vitamins, drugs(including those administered for therapeutic purposes as well as thoseadministered for illicit purposes), pollutants, pesticides, andmetabolites of or antibodies to any of the above substances. The termanalyte also includes any antigenic substances, haptens, antibodies,macromolecules, and combinations thereof. A non-exhaustive list ofexemplary analytes is set forth in U.S. Pat. No. 4,366,241, at column19, line 7 through column 26, line 42, the disclosure of which isincorporated herein by reference. Further descriptions and listings ofrepresentative analytes are found in U.S. Pat. Nos. 4,299,916;4,275,149; and 4,806,311, all incorporated herein by reference.

“Label reagent” refers to a substance comprising a detectable labelattached with a specific binding member. The attachment may be covalentor non-covalent binding, but the method of attachment is not critical tothe present invention. The label allows the label reagent to produce adetectable signal that is related to the presence of analyte in thefluid sample. The specific binding member component of the label reagentis selected to directly bind to the analyte or to indirectly bind theanalyte by means of an ancillary specific binding member, which isdescribed in greater detail hereinafter. The label reagent can beincorporated into the test device at a site upstream from the capturezone, it can be combined with the fluid sample to form a fluid solution,it can be added to the test device separately from the test sample, orit can be predeposited or reversibly immobilized at the capture zone. Inaddition, the specific binding member may be labeled before or duringthe performance of the assay by means of a suitable attachment method.

“Label” refers to any substance which is capable of producing a signalthat is detectable by visual or instrumental means. Various labelssuitable for use in the present invention include labels which producesignals through either chemical or physical means. Such labels caninclude enzymes and substrates, chromogens, catalysts, fluorescentcompounds, chemiluminescent compounds, and radioactive labels. Othersuitable labels include colloidal metallic particles such as gold,colloidal non-metallic particles such as selenium or tellurium, dyed orcolored particles such as a dyed plastic or a stained microorganism,organic polymer latex particles and liposomes, colored beads, polymermicrocapsules, sacs, erythrocytes, erythrocyte ghosts, or other vesiclescontaining directly visible substances, and the like. Typically, avisually detectable label is used as the label component of the labelreagent, thereby providing for the direct visual or instrumental readoutof the presence or amount of the analyte in the test sample without theneed for additional signal producing components at the detection sites.

The selection of a particular label is not critical to the presentinvention, but the label will be capable of generating a detectablesignal either by itself, or be instrumentally detectable, or bedetectable in conjunction with one or more additional signal producingcomponents, such as an enzyme/substrate signal producing system. Avariety of different label reagents can be formed by varying either thelabel or the specific binding member component of the label reagent; itwill be appreciated by one skilled in the art that the choice involvesconsideration of the analyte to be detected and the desired means ofdetection. As discussed below, a label may also be incorporated used ina control system for the assay.

For example, one or more signal producing components can be reacted withthe label to generate a detectable signal. If the label is an enzyme,then amplification of the detectable signal is obtained by reacting theenzyme with one or more substrates or additional enzymes and substratesto produce a detectable reaction product.

In an alternative signal producing system, the label can be afluorescent compound where no enzymatic manipulation of the label isrequired to produce the detectable signal. Fluorescent molecules such asfluorescein, phycobiliprotein, rhodamine and their derivatives andanalogs are suitable for use as labels in such a system.

The use of dyes for staining biological materials, such as proteins,carbohydrates, nucleic acids, and whole organisms is documented in theliterature. It is known that certain dyes stain particular materialspreferentially based on compatible chemistries of dye and ligand. Forexample, Coomassie Blue and Methylene Blue for proteins, periodicacid-Schiffs reagent for carbohydrates, Crystal Violet, Safranin O, andTrypan Blue for whole cell stains, ethidium bromide and Acridine Orangefor nucleic acid staining, and fluorescent stains such as rhodamine andCalcofluor White for detection by fluorescent microscopy. Furtherexamples of labels can be found in, at least, U.S. Pat. Nos. 4,695,554;4,863,875; 4,373,932; and 4,366,241, all incorporated herein byreference.

“Signal producing component” refers to any substance capable of reactingwith another assay reagent or with the analyte to produce a reactionproduct or signal that indicates the presence of the analyte and that isdetectable by visual or instrumental means. “Signal production system”,as used herein, refers to the group of assay reagents that are needed toproduce the desired reaction product or signal.

“Ancillary specific binding member” refers to any member of a specificbinding pair which is used in the assay in addition to the specificbinding members of the conjugate or capture reagent. One or moreancillary specific binding members can be used in an assay. For example,an ancillary specific binding member can be capable of binding theconjugate with the analyte of interest, in instances where the analyteitself could not directly attach to the conjugate. Optionally, theancillary binding member may be capable of binding the analyte with thecapture reagent, in instances where the analyte itself could notdirectly attach to the capture reagent. The ancillary specific bindingmember can be incorporated into the assay device or it can be added tothe device as a separate reagent solution.

“Solid phase” refers to any solid material to which analyte, analytecomplexes, or assay reagents become bound and which can be separatedfrom unreacted assay reagents, test sample, or test solutions. Forexample, the solid phase may involve beads, magnetic particles, latexparticles, test tubes, microtiter plates, or any other solid material.Generally, the solid phase is any suitable chromatographic, bibulous,porous, isotropic, or capillary material, hereinafter referred to asporous material, which forms the basis of the test device.

“Capture reagent” refers to a specific binding member that is attachedwithin or upon a portion of the solid phase to form a “capture zone”.The method of attachment is not critical to the present invention. Thecapture reagent is selected to bind the analyte, the conjugate, or acomplex thereof.

“Ancillary material” shall mean any material that may be employed in anassay in accordance with the present invention. For example, buffer willnormally be present in the labeling means, the neutralization means, aswell as stabilizers for the assay medium and assay components.Frequently, in addition to these additives, additional protein, such asalbumin, or surfactant, non-ionic or ionic, binding enhancer, forexample, polyalkylene glycol, or the like, may be present, includingfree antibody, analyte analog, or other unrelated ligand, for thepurpose of removing or adding material or to modify the amount,position, partitioning, or appearance of the analyte or other compoundused in the invention.

“Observable signal” as used herein refers to a signal produced in theclaimed devices and methods that is detectable by visual inspection.Without limitation, the type of signal produced depends on the labelreagents and marks used (described herein). Generally, observablesignals indicating the presence or absence of an analyte in a sample maybe evident of their own accord, e.g., plus or minus signs orparticularly shaped symbols, or may be evident through the comparisonwith a panel such as a color indicator panel.

“Axial flow” as used herein refers to lateral, vertical or transverseflow through a particular matrix. The type of flow contemplated in aparticular device, assay or method varies according to the structure ofthe device. Without being bound by theory, lateral, vertical ortransverse flow may refer to flow of a fluid sample from the point offluid contact on one end or side of a particular matrix to an areadownstream of this contact. The downstream area may be on the same sideor on the opposite side of the matrix from the point of fluid contact.For example, in vertical flow devices of the present invention, axialflow may progress vertically from and through a first member (top tobottom) to a second member and from there on to an absorbent medium.

“Porous material” refers to any material that may be employed in thelateral assays described herein that are opaque in a dry state andtransparent or semi-transparent in a moist state (e.g., nitrocellulose).

“Absorbent material” as used herein refers to material used in verticalflow devices and assays that allows and promotes sample flow through thefirst and second members. Such materials may be as described in, e.g.,U.S. Pat. No. 4,632,901, such as, for example, fibrous materials such ascellulose acetate fibers, cellulose or cellulose derivatives, polyester,or polyolefin. Generally, the absorbent material, as used herein, shouldmaintain direct or intimate contact with the second member in order topromote fluid flow therethrough. Contemplated absorbent materials havingfluid absorptive qualities are generally compressible and may becompressed in devices of the present invention to ensure contact withthe second member or positive control element.

“Registration means” or “indexing means” refers to physical means usefulfor placement of the first and second members in the vertical flowdevices of the present invention. These means may involve the use ofrails contained in the outer wall of a device coupled with notches orslots located in the first and second members. Without limitation, thesemeans are useful for providing an orientation of control markings inrelation to capture zones such that in the presence or absence of ananalyte of interest and detectable reagent coupled thereto, all markingsand zones may be separately distinguishable. Occasionally, these meansmay also equally apply to placement of the absorbent medium in thevertical assay devices of the present invention.

“Orientation indicia” refers to a separate marking located on a verticalflow device of the present invention that permits the user of the deviceto quickly and accurately determine the correct reading orientation ofthe device to determine a positive and/or negative result after use ofsuch device in accordance with the presently described methods.

Introduction

The present invention relates generally to devices and methods fordetection of analytes in test samples. More specifically, the presentinvention provides solid phase test strip devices and methods thatcombine an internal indicator on the test strip with an external marklocated on a support.

Test Devices

In a particular aspect, the present invention provides a test device fordetermining the presence or absence of an analyte in a fluid sample. Thetest device includes a support bearing a mark thereon, and a matrixdefining an axial flow path. Typically, the matrix further includes asample receiving zone, a label zone, and an observation area thatcontains a capture zone. In a related embodiment, the matrix furtherincludes an absorbent zone disposed downstream of the observation area.

In a preferred embodiment, the sample receiving zone accepts a fluidsample that may contain an analyte of interest. In another embodiment,the sample receiving zone is dipped into a fluid sample. A label zone islocated downstream of the sample receiving zone, and contains a mobilelabel reagent that recognizes the analyte of interest. Further, anobservation area is disposed downstream of from the label zone, andcontains an immobilized capture reagent that binds to the analyte ofinterest. Thus, as the fluid sample flows along the matrix, the analyteof interest will first bind with the mobile label reagent in the labelzone, and then bind to the immobile capture reagent in the capture zoneof the observation area. The observation area is made of a material thatis opaque in a dry state and transparent in a moist state. Thus, themark on the support is visible through the observation area when theobservation area is in the moist state.

In another preferred embodiment, the fluid sample flows along a flowpath running from the sample receiving zone (upstream), through thelabel zone (midstream), and then to the observation area (downstream).Optionally, the fluid may thereafter flow to the absorbent zone.

In a preferred embodiment, the sample receiving zone is made of anabsorbent application pad. Suitable materials for manufacturingabsorbent application pads include, but are not limited to, hydrophilicpolyethylene materials or pads, glass fiber filter paper or pads,desiccated paper, paper pulp, fabric, and the like. In a relatedembodiment, the sample receiving zone is constructed from any materialthat absorbs water.

In a preferred embodiment, the absorbent application pad is made of anymaterial from which the fluid sample can pass to the label zone.Further, the absorbent application pad may be constructed to act as afilter for cellular components, hormones, particulate, and other certainsubstances that may occur in the fluid sample. Application pad materialssuitable for use by the present invention also include those applicationpad materials disclosed in U.S. Pat. No. 5,075,078, incorporated hereinby reference.

In yet another preferred embodiment, the absorbent application pad mayincorporate other reagents such as ancillary specific binding members,fluid sample pretreatment reagents, and signal producing reagents.

In a particularly preferred embodiment, the analyte of interest is fromthe group including heartworm antigens, hCG (human chorionicgonadotropin), streptococcus A, and other bacterial antigens.

In another preferred embodiment, the test device is configured toperform an immunological analysis process. In yet another embodiment,the liquid transport along the matrix is based upon capillary action,whereby the liquid transport path can be formed not only by one or morelayers of absorbent material, for example paper or fleece, but also by agap which is sucked full by capillary action.

In a preferred embodiment, the label zone is capable of non-bibulouslateral flow. By “non-bibulous lateral flow” is meant liquid flow inwhich all of the dissolved or dispersed components of the liquid arecarried at substantially equal rates and with relatively unimpaired flowlaterally through the membrane, as opposed to preferential retention ofone or more components as would occur, e.g., in materials capable ofadsorbing or imbibing one or more components.

In a further preferred embodiment, the label zone is made of a typicalnon-bibulous material such as high density polyethylene sheet materialmanufactured by Porex Technologies Corp. of Fairburn, Ga., USA. Thesheet material has an open pore structure with a typical density, at 40%void volume, of 0.57 gm/cc and an average pore diameter of 1 to 250micrometers, the average generally being from 3 to 100 micrometers. Theoptimum pore diameter for the membrane for use in the invention is about10 to about 50 μm. The membranes typically are from about 1 mil to about15 mils in thickness, typically in the range of from 5 or 10 mils, butmay be up to 200 mils and thicker. The membrane may be backed by agenerally water impervious layer, such as mylar. When employed, thebacking is generally fastened to the membrane by an adhesive, such as 3M444 double-sided adhesive tape. Typically, a water impervious backing isused for membranes of low thickness. A wide variety of polymers may beused provided that they do not bind nonspecifically to the assaycomponents and do not interfere with flow of the sample. Illustrativepolymers include polyethylene, polypropylene, polystyrene and the like.Alternatively, the membrane may be self supporting. Other non-bibulousmembranes, such as polyvinyl chloride, polyvinyl acetate, copolymers ofvinyl acetate and vinyl chloride, polyamide, polycarbonate, polystyrene,and the like, can also be used.

In yet another preferred embodiment, the label zone is made of amaterial such as untreated paper, cellulose blends, nitrocellulose,polyester, an acrylonitrile copolymer, and the like. The label zone maybe constructed to provide either bibulous or non-bibulous flow. In anespecially preferred embodiment, the label zone is made of a nonwovenfabric such as Rayon or glass fiber. Other label zone materials suitablefor use by the present invention include those chromatographic materialsdisclosed in U.S. Pat. No. 5,075,078, which is herein incorporated byreference. In a preferred embodiment, the label zone material may betreated with labeled solution that includes blocking and stabilizingagents. Blocking agents include bovine serum albumin (BSA), methylatedBSA, casein, nonfat dry milk. Stabilizing agents are readily availableand well known in the art, and may be used, for example, to stabilizecolored labels.

In all of the above, employment of the selected blocking and stabilizingagents together with colored moieties in the labeling zone followed bythe immobilization of the blocking and stabilizing agents on the support(by, e.g., a freeze-drying process, or a forced air heat drying process)is of utmost importance for improved performance of the device. It iswell known that visible moieties, especially particles, aggregate uponair-drying and do not readily rehydrate in contact with a liquid sample.Therefore, absent conversion to the nonbibulous surface, instead ofbeing transported to the capture zone with the sample, the visiblemoieties will remain trapped in the labeling zone.

In a particularly preferred embodiment, the observation area will bemade of a material that is opaque when in a dry state, and transparentwhen in a moistened state, examples of which include nitrocellulose,nylon, and hydrophilic polyvinylidene difluoride (PVDF). Hydrophilicpolyvinylidene difluoride (PVDF) is commercially available form the firmMillipore, Bedford, U.S.A. under trademark Immobilon AV. However, on thebasis of the present description, the expert can also select othermaterials and especially synthetic material membranes which fulfill theabove-mentioned conditions. It is believed that the refractive index ofthe synthetic material is of major influence to this characteristic. Itis to be assumed that porous materials, the refractive index of which isclose to that of the sample liquid, have the property of becomingtransparent in a moist state.

In a particularly preferred embodiment, the observation area is anitrocellulose web assembly made of Millipore nitrocellulose rolllaminated to a clear Mylar backing. In another embodiment, theobservation area is made of nylon.

Those skilled in the art will appreciate that various methods could beused to test the desired property of the observation area, and whetherit is sufficiently opaque in the dry state, and sufficiently transparentin the moist state. For example, one could put a mark on a piece ofplastic, and then place the test strip on top of the plastic andvisually determine if it is opaque or transparent. Other methods includethe use of scanner devices, or other means for determining opticaldensity of the apparent line.

The artisan will appreciate the whole of the observation area, or only aportion thereof, may become transparent when moistened. In eitherembodiment, the portion of the observation area that is disposed abovethe mark should become transparent when moistened. In a relatedembodiment, the observation area will contain one or more distinct areasthat are opaque in the dry state, and transparent in the moist state.

In a preferred embodiment, the capture zone may be constructed from anyof the materials as listed above for the observation zone. In aparticularly preferred embodiment, the capture zone is made of the samematerial as the observation zone. The artisan will recognize that thepresent invention envisages a test device with one or more capturezones.

Further embodiments include capture zones that include microporousmaterials made from nitrocellulose, by which term is meant any nitricacid ester of cellulose. Thus suitable materials may includenitrocellulose in combination with carboxylic acid esters of cellulose.The pore size of nitrocellulose membranes may vary widely, but ispreferably within 5 to 20 microns, preferably 8 to 15 microns. Toprovide non-bibulous flow, these materials may be treated with blockingagents that can block the forces which account for the bibulous natureof bibulous membranes. Suitable blocking agents include bovine serumalbumin, methylated bovine serum albumin, whole animal serum, casein,and non-fat dry milk.

In a preferred embodiment, the observation area further includes aprocedural control line, to verify that the sample flow is as expected.The control line is a spatially distinct region that includes animmobilized binding member which reacts with a labeled reagent. In apreferred embodiment, the procedural control line contains an authenticsample of the analyte of interest, or a fragment thereof. In anotherpreferred embodiment, the control line contains antibody that isspecific for, or otherwise provides for the immobilization of, thelabeled reagent. In operation, a labeled reagent binds to the controlline, even when the analyte of interest is absent from the test sample.

In a related embodiment, a control conjugate is introduced into the flowsample upstream from the control line. For example, the controlconjugate may be added to the fluid sample before the sample is appliedto the assay device. Alternatively, the control conjugate may bediffusively bound in the sample receiving zone, or in the label zone.

In a preferred embodiment, the control conjugate includes a controllabel and a control reagent. Typically, a control reagent is chosen tobe different from the reagent that is recognized by the capture reagent.Further, the control agent is generally not specific for the analyte. Ina preferred embodiment, the control reagent binds to a control capturepartner that is immobilized on the procedural control line. Thus thecontrol conjugate is directly detected in the control line.

In yet another embodiment, the detectable control label is the samelabel used for the label reagent. In a preferred embodiment, the controllabel is different from the test label, so that the user can more easilydetermine the results of the assay. In a particularly preferredembodiment, the control label and the test label include colored beads,where the control and test beads are of different colors.

In a preferred embodiment, the control label includes streptavidin, andthe control capture partner includes biotin, which couples to the avidinspecifically. In a particularly preferred embodiment, the control labelincludes biotin, and the control capture partner includes streptavidin.The artisan will appreciate that other “irrelevant” binding pairs canalso be used-such as antigen/antibody reactions unrelated to analyte.

The use of a control line is helpful in that appearance of a signal inthe control line indicates the time at which the test result can beread, even for a negative result. Thus, when the expected signal appearsin the control line, the presence or absence of a signal in the capturezone can be noted.

In another preferred embodiment, the matrix may further incorporate anabsorbent zone. The absorbent zone can act to increase the amount offluid sample that travels through the capture zone.

In this embodiment, the absorbent zone is located downstream from thecapture zone and can be a means for removing excess sample and freelabel other than the analyte of interest from the matrix of the device.Generally, the absorbent zone will consist of an absorbent material suchas filter paper, a glass fiber filter, or the like.

In a preferred embodiment, the device may also contain an end of assaycontrol zone indicator. The control zone indicator may consist of a pHindicating reagent (such as bromocresol green) impregnated in theabsorbent zone or at a location downstream of the capture zone. Uponcontact with the sample, a pH change occurs in the processed matrix.This pH shift converts the pH indicator to a different color (forinstance, bromocresol green may be converted from yellow to blue) whichis seen in an observation window over the control zone. This technologymay also serve as an internal assay control.

In a related embodiment, the end of assay control zone may beconstructed by applying a line of soluble ink on the capture zone (atthe interface with the absorbent zone). The liquid front moving throughthe capture zone will solubilize the ink and transfer it into theabsorbent. The resulting color change will be seen in an observationwindow above the absorbent zone, signifying end of assay.

In a preferred embodiment, the capture reagent binds with the analytefor the completion of a sandwich complex. The capture reagent can bechosen to directly bind the analyte or indirectly bind the analyte bybinding with an ancillary specific binding member which is bound to theanalyte. In addition, the capture reagent may be immobilized on thesolid phase before or during the performance of the assay by means ofany suitable attachment method. Typically, the capture site of thepresent invention is a delimited or defined portion of the solid phasesuch that the specific binding reaction of the capture reagent andanalyte is localized or concentrated in a limited site, therebyfacilitating the detection of label that is immobilized at the capturesite in contrast to other portions of the solid phase. In a relatedembodiment, the capture reagent can be applied to the solid phase bydipping, inscribing with a pen, dispensing through a capillary tube, orthrough the use of reagent jet-printing or other techniques. Inaddition, the capture zone can be marked, for example with a dye, suchthat the position of the capture zone upon the solid phase can bevisually or instrumentally determined even when there is no labelimmobilized at the site.

Those of skill in the art will recognize that a variety of direct andindirect assay formats may be employed in the present invention. In apreferred embodiment, a direct assay format is used. Direct assays areexemplified by those that detect the presence of an antigen in a sample,as well as those that detect the presence of an antibody in a sample. Adirect assay that detects the presence or absence of an antigen in asample generally includes a label zone that contains at least oneantibody label reagent that is specific for the antigen. In a preferredembodiment, the antibody is coated on colored beads. Likewise, a directassay that detects the presence or absence of an antigen in a samplegenerally includes a label zone that contains at least one antigen thatis reactive with an antibody-analyte of interest. In this embodiment,the antigen is coated on colored beads.

As provided above, particular devices of the present invention include asupport. The support in these devices provides a convenient platform forperformance of the assay. However, the composition and shape of thesupport are not critical and may vary. Occasionally, the support may becomprised of a plastic or nylon material.

In another embodiment the present devices may be in the form of adipstick. Generally, dipsticks of the present invention are functionallyanalogous to the lateral assays described herein excepting the method ofcontacting a fluid sample. In embodiments configured as a dipstick, thematrix and support will generally be located on one end of the dipstick.The configuration of such devices will allow the device to be dipped orcontacted with a fluid sample with one end of a matrix that is analogouswith the sample receiving zone in lateral assays and devices alsodescribed herein. After contacting the fluid sample, the samplepreferably migrates in an axial flow path through the matrix from thesample receiving zone to the label zones and observation areas.Alternatively, the devices of the present invention may be shaped sothat samples may be applied to the device by means other than dipping,e.g., application of controlled amounts of sample by pipettes or thelike.

In a preferred embodiment, the result displayed by the test deviceincorporates a symbol provided by the mark on the bottom support. Due tothe transparent nature of the observation area, the mark on the supportis detectable through the moistened observation area. Thus, the shape ofthe mark will be displayed regardless of whether the analyte of interestis or is not present in the fluid sample. Typically, the shape of themark provides a first symbol to represent a negative result. In arelated embodiment, the symbol provided by the mark on the support is aminus (−) symbol. In a particularly preferred embodiment, the mark willbe visible to the unaided eye, although those of skill in the art willappreciate that a variety of detection approaches may be employed. Inanother preferred embodiment, the mark will be blue or red, or any colorwhich provides contrast with the surrounding area, which is typically alighter color, such as white.

In a preferred embodiment, the mark on the support will operate inconcert with a symbol provided by the capture zone. When the desiredanalyte is present in the fluid sample, label is collected at thecapture zone. The shape of the labeled capture zone is such that, incombination with the mark, a second symbol is provided representing apositive result. In a related embodiment, the symbol presented by thiscombination is a plus (+) symbol.

In yet another preferred embodiment, the capture zone has a shape suchthat, independent of any symbol provided by that mark on the substrate,labeled reagent at the capture zone will form a second symbol torepresent a positive test result.

In a preferred embodiment, the mark on the support is oriented so thatthe longitudinal axis of the mark is aligned with the direction of thesample flow, while the longitudinal axis of the capture zone isperpendicular to the direction of the sample flow. In a relatedembodiment, these axes may be at oblique angles to the sample flow. Inyet another related embodiment, these axes may be oriented at any angleto the sample flow, so long as the axis of the mark and the axis of thecapture zone are substantially perpendicular to each other.

In another preferred embodiment, the mark on the support includes ashape such that, in the presence or absence of the analyte of interestin the fluid sample, a logo is displayed in the observation area. In arelated embodiment, the marked support provides one or more predefinedsymbols to be displayed when the observation area is moistened. Thesesymbols may be read in connection with, or independent from, any signalthat may be provided by the capture zone.

In yet another embodiment, the mark may be displayed below the capturezone, but not necessarily on the bottom support. For example, the markmay be contained on the Mylar strip underlying the nitrocellulose matrixmembrane. Alternatively, the mark may be contained on an intermediatemember disposed between the test strip and the bottom support. In apreferred embodiment, the intermediate member is a plastic stripdisplaying the mark. The member may or may not be affixed to either thetest strip or the bottom support. In a preferred embodiment, theintermediate member is configured to remain in place once the testapparatus is constructed. In a related embodiment, the mark is applied,for example, by an embossing, printing, or stamping technique.

In still another preferred embodiment, the observation area includes anegative control area. The purpose of this control area is to alert theuser that the test device is not working properly. In a preferredembodiment, the negative control is that part of the observation areaoutside of the capture zone, and does not include any part of theobservation area located directly above the mark on the support. Whenworking properly, no signal or mark should be visible in the negativecontrol area.

In a preferred embodiment, the test device includes a hollow casing orhousing having an application aperture and an observation port. In thisembodiment, the flow matrix is contained within the hollow casing, andthe fluid sample is added to the matrix through the aperture, which isan opening located in an upstream location on the housing.

Typically, the aperture is located above the sample application pad. Ina related embodiment, an aperture may be disposed in any location abovethe matrix that would provide for facile addition of fluid sample orreagent to the matrix.

To detect the result, the observation area may be examined through theport. The artisan will appreciate that an observation port may bedisposed in any location above the matrix that would provide for thedetection of a signal event, whether the signal comes from theobservation area, the absorbent zone, or elsewhere. In a preferredembodiment, the port is located above the observation area. Generally,the observation port is open, or the port may be covered by atransparent covering such as glass or plastic.

From the foregoing, it is appreciated that the outer casing or housingof the device may take various forms. Typically, it will include anelongate casing and may have a plurality of interfitting parts. In aparticularly preferred embodiment, the housing includes a top cover anda bottom support. In a preferred embodiment, the bottom support displaysa mark thereon, and in a related embodiment, the top cover contains anapplication aperture and an observation port. In another embodiment, thehousing may also contain dividers between the matrix strips to inhibitflow of fluid sample between strips.

In a preferred embodiment, the housing is made of moisture impervioussolid material, for example, a plastic material. It is contemplated thata variety of commercially available plastics, including, but not limitedto, vinyl, nylon, polyvinyl chloride, polypropylene, polystyrene,polyethylene, polycarbonates, polysulfanes, polyesters, urethanes, andepoxies maybe used to construct a housing. The housing may be preparedby conventional methodologies, such as standard molding technologiesthat are well known and used in the art. The housing may be produced bymolding technologies which include, but are not limited to, injectionmolding, compression molding, transfer molding, blow molding, extrusionmolding, foam molding, and thermoform molding. The aforementionedmolding technologies are well known in the art and so are not discussedin detail herein. See for example, Processes And Materials OfManufacture, Third Edition, R. A. Lindsberg (1983) Allyn and Baron pp.393–431.

It will be appreciated by one skilled in the art that a test stripdevice can be made of more than one material (e.g., different zones orsites can be made of different materials) and a flow-through device canhave more than one layer, wherein different layers can be made ofdifferent materials, so long as the multiple materials or layers are influid-flow contact with one another thereby enabling the passage of testsample between the materials or layers. Fluid-flow contact permits thepassage of at least some components of the test sample between the zonesor layers of the device. Fluid-flow is preferably uniform along thecontact interface between the different zones or layers. The presentinvention further provides for different reagents disposed on differentmaterials, and different reagents disposed on different zones. Forexample, the specific binding member contained on a certain material inthe label zone may or may not be the same binding member that iscontained on a different material in the capture zone.

In further preferred embodiments, the test device of the presentinvention can have many configurations, several of which are dependentupon the material chosen for the solid phase. For example, the testdevice can include a solid phase material configured for use in alayered flow-through assay device, a chromatographic column, a dipstickor a test strip.

In a second aspect, the present invention provides a method fordetermining the presence or absence of one or more analytes in a fluidsample. The assay method includes providing a test device as describedabove, and administering a fluid sample to the test device. Byinspecting the observation area of the test device, the user is able todetermine whether the fluid sample contains the one or more analytes ofinterest. Typically, this is accomplished by observing either a plus (+)or minus (−) signal in the observation area for each analyte ofinterest.

In a third aspect, the present invention provides a kit for determiningthe presence or absence of an analyte in a fluid sample. The assay kitof the present invention will include containers for holding anynecessary reagent, test membrane, sample applicator, filter, and thelike, as well as instructions which set forth a protocol for practicingthe method of use as described above. Typically, the components of thekit will be present in a suitable package containing sufficientcomponents for performing one or more assays.

The present invention is particularly suitable for a test device asshown in the accompanying drawings, and described in detail as follows.It is understood that the drawings are provided for purposes ofillustration and not meant limit the scope of the present invention.

FIG. 1 shows a first embodiment of a test device 10 constructed inaccordance with the teachings of the present invention.

Test device 10 has a bottom support 14, a flow matrix 18, a top cover22, and a desiccant 26. In its longitudinal direction, matrix 18 can besubdivided into a sample application zone 30, a label zone 34, anobservation area 38, and an absorbent zone 42.

The bottom support further includes a mark 46 thereon. The mark isdisposed beneath observation area 38. The sample application zone islocated at an upstream location on matrix 18, and is configured toreceive the fluid sample. Label zone 34 is located downstream ofapplication zone 30, and contains label reagent. The observation area islocated downstream of the label zone, and includes a capture zone 40that contains capture reagent. Absorbent pad 42 is located downstream ofobservation area 38.

Top cover 22 has an application aperture 48 disposed above the sampleapplication pad, and an observation port 52 disposed above theobservation area. In cooperation, the top cover and the bottom supportare configured to provide a housing for matrix 18 and desiccant 26. Asshown, the desiccant is typically positioned separately from the matrix.

Prior to use, when in the dry state, the observation area is opaque, andthus no signal is manifested through the observation port.

FIG. 2 illustrates the test device of the present invention showing anegative test result, in the instance where the fluid sample does notcontain the analyte of interest.

In one operative embodiment, the sample fluid is added through aperture48, and on to application pad 30. The fluid sample is transported fromapplication pad 30 to label zone 34, where the fluid is allowed to reactwith labeled reagent. Labeled reagent then binds with any analyte ofinterest contained in the fluid sample.

Next, the fluid sample is advanced to observation area 38, and then onto the absorbent zone. Observation area 38, now moistened by the samplefluid, becomes transparent. Consequently, mark 46 becomes visiblethrough observation port 52. Through the observation port, the user canthen view the mark displayed on the bottom support.

As shown here, there is no analyte present, and consequently no label isbound at capture zone 40. The resulting signal as manifested throughobservation port 52 is therefore in the shape of a minus (−) sign.

FIG. 3 illustrates the test device of the present invention showing apositive test result, in the instance where the fluid sample containsthe analyte of interest.

In operation, the sample fluid is added through aperture 48, and on toapplication pad 30. The fluid sample is transported from application pad30 to label zone 34, where the fluid is allowed to react with labeledreagent. Labeled reagent then binds with the analyte of interestcontained in the fluid sample. Next, the fluid sample is advanced toobservation area 38, and labeled analyte is restrained by capturereagent immobilized in capture zone 40. Unbound material in the fluidsample continues to flow into absorbent zone 42.

Observation area 38, now moistened by the sample fluid, becomestransparent. Consequently, mark 46 becomes visible through observationport 52. Through the observation port, the user can then view the markdisplayed on the bottom support, in concert with the signal provided bythe label contained in the capture zone. The resulting signal asmanifested through observation port 52 is in the shape of a plus (+)sign.

FIG. 4 shows the test device of the present invention, where observationarea 38 further includes a procedural control line 56 that develops withany fluid sample flow, whether or not analyte is present in the sample.

Vertical Flow Devices

In a further embodiment, the device of the invention may alternativelycomprise a vertical flow device. In such devices, as a first member, aporous member may be provided, such as a membrane or filter, to which acapture reagent may be immobilized thereon. The first member preferablyfurther comprises an observation area comprising a defined capture zoneand is preferably comprised of a material that is opaque in a dry stateand transparent in a moist state. The first member provides for fluidflow therethrough, from the top to the bottom surfaces. Similar to thecapture reagents described above, preferred reagents are capable ofrestraining an analyte of interest on the porous first member. Thesecapture reagents are useful in that they are capable of separatinganalyte from the fluid sample being analyzed.

In another aspect, the device may further comprise, as a second member,an absorbent member having fluid pathways therethrough from its upper tolower surfaces. The second member is generally disposed below, and influid flow contact with, the first member in the present devices and isselected to allow fluid flow through the first member when a fluidsample is introduced to the upper surface of the first member. In oneaspect, the second member comprises a porous positive control membranesupport. In a related aspect, the second member may act to support thefirst member and also contains a positive control zone. The uppersurface of the first member is merely the surface lying away from thesurface contacting or otherwise in communication with the second member.

In another aspect, vertical flow type devices of the present disclosureinclude an optional absorbent medium. Generally, the absorbent medium isuseful for absorbing liquid sample, labeled reagent and/or washingsolution after flow through the first and second members. The absorptioncapacity of contemplated absorbent mediums of the present invention areuseful for promoting fluid flow through both the first and secondmembers and thus avoiding stagnant fluid remaining on these members. Toaccomplish this function, the absorbent medium is preferably in close orintimate contact with the second member or positive control element. Inone aspect, the absorbent medium may be compressed in a device, belowthe second member.

The above device may be utilized through the contact or introduction ofa fluid sample suspected of containing one or more analytes and thenallowing the sample to migrate through the first and second members,whereby the one or more analytes bind to one or more capture reagentsbound to the first member. After the sample has migrated through bothmembers one or more labeled reagents (described above) may be introducedto the upper surface of the first member and allowed to migrate throughthe first and second members. The labeled reagent is selected to bind toone or more of the analytes suspected of being present in the sample.Thereafter, a washing step may be performed to wash unbound labeledreagent from the first and second members. The presence or absence of aparticular analyte may then be detected by visually inspecting the firstmember to locate marks, logos, symbols, colors, etc., which indicate apositive or negative result corresponding to the presence or absence ofparticular analytes in a given sample.

Generally, marks indicating a negative result may be disposed on thesecond member which may become visible through the first member when thefirst member is in a moist state (i.e., any time after introduction ofthe sample to the device). Positive results indicating the presence ofone or more analytes in a sample are evident through an indication thatthe labeled reagent has bound to a particular analyte, i.e., thegeneration of a positive signal. The first member is generally comprisedof a material described above that is opaque in a dry state andtransparent in a moist state.

Suitable washing reagents may be, for example, buffers, solutionscontaining detergents, defined ionic strength chelators, proteins orother components useful for optimizing separation of bound and freelabel. Suitable washing reagents may have varying characteristics,however they should not interfere with or otherwise degrade: (1) theanalytes or reagents themselves, (2) the interaction of the analyte withthe binding or label reagents, or (3) the signal generated indicatingthe presence or absence of an analyte as a result of utilizing theclaimed devices or methods.

FIG. 5 shows a top view of a vertical flow device. The device sidewall(61), orientation indicia (63), guide rails (62) and center chamber (64)are shown. In one aspect, liquid samples may be poured or otherwiseintroduced into the center chamber (64) that is surrounded by the devicesidewall (61). The device guide rails (62) are useful for providingorientation, registration or indexing capability for additionalcomponents, for example, for positioning the porous positive controlmembrane support (see FIG. 6 (70)) (the “second member”) andfilter/membrane (see FIG. 7 (71)) containing a capture/test zone (68)(together, the “first member”) within the device. In one embodiment, thedevice contains one guide rail. In a related embodiment, the device maycontain one or more guide rails, for example between about 1 to about 50or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, about 15, about20, about 25 about 30, about 35, about 40, about 45, about 50; or aseries of means for positioning the porous positive control membranesupport and filter/membrane containing a capture/test zone within thedevice such, as a series of ridges or notches. In another aspect, thepresent vertical flow device may optionally contain an orientationindicia (63) useful for positioning the positive control (66) andcapture/test zones (68) in a fashion such that when a positive testresult is formed the positive control line or lines (66) and thecapture/test zone or zones (68) are visible in a predeterminedorientation. Such predetermined orientations may comprise a reference tothe orientation indicia to determine the orientation of a positivecontrol line and/or a test/capture zone. For example, the positivecontrol line may be oriented horizontally and the test zone may beoriented vertically; without limitation, both lines may be inperpendicular relation to one another. In one embodiment a negative testresult comprises the formation of a minus symbol (−) as provided in FIG.8 (66), in a related embodiment a positive test result comprises theformation of a plus symbol (+) FIG. 9 (66 & 69).

In one aspect, a porous, positive control-support for the porous filteror membrane (FIG. 2 (70)) is positioned in a device below the porousfilter or membrane (FIG. 3 (71)). Generally, the porous, positivecontrol-support for the porous filter or membrane is positioned belowthe porous filter or membrane with respect to the direction of sampleintroduction (FIG. 10 (70 & 71)). In one aspect, these elements may be“notched” (67) in accordance with the above description to complementthe guide rails (62), or means for positioning, provided in the device.In another aspect, the control line (66) is printed onto apositive-control support (65) and guide rails (62) orient this line orelement in a predetermined orientation in the device (e.g.,horizontally). In a related aspect, this line may become the horizontalcomponent of the “plus” sign formed in a positive test result (FIG. 9)or the horizontal “minus” sign provided in a negative test result (FIG.8). Such lines or elements may be made by standard printing or othermethods described above.

In one aspect, prior to use, the positive control line (66) is invisibleto the naked eye, due to coverage by an opaque dry membrane (69). In oneembodiment, after a test is performed through the introduction of aliquid sample, the opaque membrane becomes semi-or fully-transparent,permitting visualization of a positive control line (66). Generally, thepositive control line is preferably visible after sample is added,regardless of whether the test result is positive or negative.

In another aspect, the porous semi-transparent when-wet, porous filteror membrane is notched (67) to fit guide rail or rails (62) orpositioning means built into the device sidewall (see FIG. 5, FIG. 7).In a further aspect, a vertical incipient, test zone (68) in the formof, e.g., a line containing capture antibody is present on the newmembrane. Prior to introduction of a “positive” sample this line (68)should not be visible to the naked eye. This line becomes the secondcomponent (69) of the “plus” sign seen in a positive test result (FIG.9). The test zone (68) can be made by standard methods used to createimmunoassay devices. Such materials and methods are described herein andmay be generally known in the art. After a test is performed, if analyteis present that corresponds to the capture reagent located in the zone,a signal is generated in the test zone or line creating a positive testresult (69). If the sample does not contain such analyte, a visible testzone is not visible (68). In a particular aspect, the formation of avisible test zone (69), after introduction of a sample containing ananalyte of interest, may be taken together with the visibility of acontrol line (66) as indicia of a positive test result.

A cross-section of an example vertical flow device is provided in FIG.10. The device sidewalls (61) in the illustrated device are vertical,however, they may optionally be positioned at an angle sloping inward.For example, the device sidewalls may slope in from the top portion ofthe well (73) pictured in FIG. 10 down to the top portion of thefilter/membrane element (70). The example vertical flow device picturedin FIG. 10 describes the device well (73) where sample is introduced,the filter/membrane elements (70), the positive control elements (71),stop elements (75), absorbent medium (72) and bottom plug (74). In oneaspect, this device may feature a new constriction (75) or other meansto vertically position the test membrane (70) and its support (71) inthe device. Such constriction or stop element (75) may be useful toensure uniform contact and level placement of the filter/membraneelements (70) and positive control elements (71). Stop elements may beconveniently molded into the outer wall (61), in the form of a shelf orother integrated leveling means, of the presently described verticalflow devices. Alternatively, the device may be assembled by firstplacing the absorbent (72) in the device (for example, from the bottomor from the top of the device) and by placing the membrane (70) and itspositive control support (71) directly on the absorbent (72). Regardlessof the method used, in this example it is frequently necessary tomaintain direct fluid flow contact between all pictured layers to assureconstant flow of liquid from the top chamber into the absorbent medium.

The invention can be better understood by way of the following exampleswhich are representative of the preferred embodiments, but are not to beconstrued as limiting the scope of the invention.

EXAMPLES Example 1

A lateral flow test device was constructed according to principles ofthe present invention. The test device included a G-III plastic bottom(#Z0846500), a G-III plastic scanner top (#Z0440900), a sample padcontaining stock assay reagent, a label pad containing stock assayreagent, an observation zone as described below, and an absorbent pad(#0841000).

A nitrocellulose web assembly membrane from Millipore, trade nameHighflow Plus Membrane, was combined with a clear Mylar backing.

A dark line was made on the inside of the plastic bottom, positionedparallel to sample flow in approximately the same area as would beunderneath the fixed minus line from a current assay strip.

This bottom was then assembled with an experimental G-IV assay strip asdescribed above, and a plastic scanner top. When a liquid sample wasadded to the test unit, the dark line, which was barely visible throughthe dry strip, became clearly visible. The results are shown generallyin FIG. 2.

Example 2

Goat anti-alpha hCG antibodies were immobilized on Hi-flow plusnitrocellulose membrane (nitrocellulose membrane cast on a transparentnylon sheet), manufactured by Millipore Inc. A second, unrelated proteinwas immobilized on the membrane for the procedural control line. Themembrane was then blocked with a protein solution and dried prior toassembly.

A sample pad (non-woven rayon fiber backed with mylar) was impregnatedwith a buffered protein solution and dried.

The label pad (non-woven rayon fiber backed with mylar) was impregnatedwith a solution containing red colored polystyrene microspheres coatedwith anti-beta hCG monoclonal antibodies, blue colored polystyrenemicrospheres coated with a binding pair member to the control lineprotein, and stabilizing agents followed by a drying process.

The sample and label pad, the capture membrane and an absorbent pad werethen assembled into a test strip similar to FIG. 4.

A visible line was printed onto the bottom half of the plastic housingof a test cassette. The line was placed directly beneath andperpendicular to the immobilized goat anti-alpha hCG test line on thecapture membrane. The preprinted line was approximately 1 mm thick and5–9 mm long. The assembled test strip was then placed on top of thepreprinted line into the bottom of the plastic housing as indicated inFIGS. 1–4, and the cassette was closed with the top portion of theplastic housing. Upon assembly, the preprinted line on the plastichousing was nearly invisible.

Upon addition of a liquid sample, the sample pad and label pad werere-hydrated and the sample wet the nitrocellulose capture membrane thusmaking it transparent. The pre-printed line which was barely visible inthe dry state was now clearly visible. The results are shown generallyin FIG. 4.

Numerous modifications may be made to the foregoing systems withoutdeparting from the basic teachings thereof. Although the presentinvention has been described in substantial detail with reference to oneor more specific embodiments, those of skill in the art will recognizethat changes may be made to the embodiments specifically disclosed inthis application, yet these modifications and improvements are withinthe scope and spirit of the invention, as set forth in the claims whichfollow. All publications or patent documents cited in this specificationare incorporated herein by reference as if each such publication ordocument was specifically and individually indicated to be incorporatedherein by reference.

Citation of the above publications or documents is not intended as anadmission that any of the foregoing is pertinent prior art, nor does itconstitute any admission as to the contents or date of thesepublications or documents.

1. A test device for determining the presence or absence of an analytein a fluid sample, the test device comprising: a) a matrix, the matrixcomprising: i) a porous first member comprising an observation areacomprising a capture zone said capture zone having an immobilizedcapture reagent that restrains the analyte, wherein the first membercomprises a material that is opaque in a dry state and transparent in amoist state; and ii) a porous second member bearing a mark thereon,disposed below and contacting said first member, wherein said secondmember allows fluid flow through the first member into and through thesecond member when a fluid sample is introduced; and b) the mark on thesecond member is detectable through the observation area in the firstmember when the first member is in the moist state; and c) wherein uponintroduction of a separate detectable label reagent that binds with theanalyte in the fluid sample, the capture zone becomes detectable in theobservation area, and wherein the detectable mark and the detectablecapture zone are separately distinguishable.
 2. The test device of claim1, wherein: a) the mark has a shape such that, when the first member isin the moist state, a first symbol is displayed to represent a negativeresult, and b) the capture zone having a shape such that, taken togetherwith the first symbol in the presence of the analyte in the sample, asecond symbol is formed to represent a positive result.
 3. The testdevice of claim 1, wherein: a) the mark has a shape such that, when thefirst member is in the moist state, a minus symbol is displayed torepresent a negative result, and b) the capture zone having a shape suchthat, taken together with the minus symbol in the presence of theanalyte in the sample, a plus symbol is formed to represent a positiveresult.
 4. The test device of claim 1, wherein the mark has a shape suchthat, in the presence or absence of the analyte in the sample, a logo isdisplayed.
 5. The test device of claim 1, wherein the mark has a shapesuch that, in the presence or absence of the analyte in the sample, oneor more predefined symbols are displayed.
 6. The test device of claim 1,wherein the first member further comprises a negative control area. 7.The test device of claim 1, wherein the wherein the label reagents arecolored label reagents and the first member further comprises aquantitative control area defining the color intensity of a given amountof analyte, wherein a higher concentration of analyte corresponds to ahigher color intensity.
 8. A test device for determining the presence orabsence of one or more analytes in a fluid sample comprising the testdevice of claim 1 further comprising a) said observation area furthercomprising more than one separately distinguishable capture zone, eachcapture zone having an immobilized capture reagent that binds withanalytes in the sample, b) said porous second member further comprisingmore than one separately distinguishable mark that are visible throughthe observation area of the first member when the first member is in themoist state; and c) wherein upon introduction of separate detectablelabel reagent that binds with analyte in the fluid sample, the capturezones become detectable though the observation area, and wherein thedetectable marks and the corresponding detectable capture zones areseparately distinguishable.
 9. The test device of claim 8, wherein: a)each of the marks have a shape such that, when the first member is inthe moist state, first symbols are displayed to represent negativeresults, and b) each of the capture zones have a shape such that, takentogether with the first symbols, in the presence of one or more analytesin the sample, second symbols are formed to represent a positive resultfor each of the analytes.
 10. The test device of claim 8, wherein: a)each of the marks have a shape such that, when the first member is inthe moist state, minus symbols are displayed to represent negativeresults, and b) each of the capture zones have a shape such that, takentogether with the minus symbols in the presence of one or more analytesin the sample, plus symbols are formed to represent positive results.11. The test device of claim 8, wherein the marks have a shape suchthat, in the presence or absence of one or more analytes in the sample,one or more logos are displayed.
 12. The test device of claim 8, whereinthe marks are shaped such that, in the presence or absence of one ormore analytes in the sample, one or more predefined symbols aredisplayed.
 13. The test device of claim 8, wherein the first memberfurther comprises a negative control area.
 14. The test device of claim8, wherein the label reagents are colored label reagents and the firstmember further comprises a quantitative control area defining the colorintensity of a given amount of analyte, wherein a higher concentrationof analyte corresponds to a higher color intensity.
 15. The test deviceof claim 1, wherein the device further comprises an absorbent mediumdisposed below but in contact with the second member.
 16. The testdevice of claim 1, wherein the device further comprises a means toorient the capture zone in relation to the mark such that, in thepresence of analyte, both the capture zone, including labeled reagentbound to the analyte, and the mark are separately distinguishable. 17.The test device of claim 16, wherein, in the presence of analyte, themark and the capture zone together form an interactive symbol indicatinga positive test result.
 18. A method of determining the presence orabsence of an analyte in a fluid sample, comprising the steps of: a)providing the test device of claim 1; b) contacting the fluid samplewith the upper surface of the first member and allowing the sample tomigrate through the first and second members, whereby the samplemoistens the first member rendering the mark detectable, and analyte, ifpresent, binds to the capture reagent in the first member; c)introducing a detectable label reagent to the upper surface of the firstmember, wherein said label reagent binds with the analyte within thecapture zone rendering the capture zone detectable; d) washing unboundlabeled reagent; and e) detecting analyte bound on the first member byinspecting the observation area of the first member, wherein thedetectable mark and the corresponding detectable capture zone areseparately distinguishable.
 19. A method of determining the presence orabsence of more than one analyte in a fluid sample, comprising the stepsof: a) providing the test device of claim 8; b) contacting the fluidsample with the upper surface of the first member and allowing thesample to migrate through the first and second members, whereby thesample moistens the first member rendering each of the marks detectable,and each analyte, if present, binds to a capture reagent in the firstmember; c) introducing a detectable label reagent to the upper surfaceof the first member, wherein said label reagent binds with each analytewithin the capture zone rendering the capture zone detectable; d)washing unbound labeled reagent; and e) detecting each analyte bound onthe first member by inspecting the observation area of the first member,wherein each detectable mark and each corresponding detectable capturezone are separately distinguishable.